Methods of Use of Biomaterial and Injectable Implant Containing Biomaterial

ABSTRACT

This invention relates to the use of a biomaterial for the treatment, repair and/or enhancement of bodily tissue insufficiencies of the vocal chords, muscles, ligaments, cartilage, post-operative regions, sexual organs and/or weight supporting areas of the feet as well as other conditions of the bones and joints. The biomaterial for use in the invention may comprise an injectable bioresorbable polysaccharide composition wherein the polysaccharide may be succinochitosan glutamate. This invention also relates to the use of a biomaterial comprising an injectable bioresorbable polysaccharide composition in which resorbable particles may be in suspension, the said particles comprising or consisting essentially of chitin and/or chitosan, which may be free of any additional formulation modifying agents, and a process for manufacturing the same. The invention also includes the use of various medicaments in the biomaterial formulations to enhance the treatment of the affected area of the body.

This application is a divisional of application Ser. No. 11/677,319filed Feb. 21, 2007, the contents of which are incorporated in theirentirety

FIELD OF INVENTION

The present invention relates to the field of biomaterials forimplantation in the human or animal body. More particularly, the presentinvention relates to methods of use of an implantable biomaterial, whichmay comprise chitin and/or chitosan. The biomaterial of this inventionmay be in the form of a gel, and may be injected, in particular by thesubcutaneous or intradermal route, to form an implant. This implant hasthe benefit of being bioresorbable.

BACKGROUND OF INVENTION

Experts in the field are familiar with various injectable implants. Forexample, silicon gels (or silicon oils) are well-known, but these gelshave the inconvenience of not being biodegradable. Moreover, silicon isoften the cause of chronic inflammation, granuloma formation and evendelayed allergic reactions. Collagen suspensions have also been verywidely used over the past ten years. However, collagen generally is ofbovine origin, which is undesirable for health and generally subject toadditional regulatory requirements. Attempts to re-implant fatty cellsremoved from the patients themselves are also reported. However, theduration of the filling effect is generally less than the patient wouldlike.

Other implants have been used, comprising a gelatine or collagensolution including, in suspension, polymethyl methacrylate (PMMA)microspheres having a diameter of 20 to 40 μm. PMMA, however, is notbiodegradable and the gelatine or collagen solution is generally derivedfrom bovine sources.

EP 0 969 883 describes an implantable gel including L-PLA (polylacticacid) microspheres with a diameter of 20 to 40 μm suspended in a carboxymethylcellulose gel (CMC). This gel is injectable and can be supplied ina sterile syringe. This product shows an acceptable efficacy but maypresent poor syringability (clogging of the required low-diameterneedles may be noted) and a biodegradability which is too slow for someof the desired applications. The particles have the tendency toaggregate in the packaging, in particular in a syringe, makinginjections difficult and leading to inconsistent results.Non-homogeneous distribution of the particles in the injection area mayactually be observed in patients. The expected aesthetic result istherefore not achieved and areas overloaded with particles are noted,sometimes adjacent to areas free of particles. The very long resorptiontime of the PLA (having a high molecular weight) may be of severalyears, which may also lead to inflammatory reactions in the long run.

There are numerous novel applications for biomaterials which do not havethe disadvantages of the prior art materials, and particularly methodsof using biomaterials which are useful as immediate filler materials,able to generate fibrosis and also capable of being resorbed to avoidchronic inflammatory reactions or rejection in the long run.

SUMMARY OF INVENTION

The present invention is directed to several new uses of biomaterialsincluding for the treatment, repair and/or enhancement of bodily tissueinsufficiencies of the vocal chords, muscles, ligaments, cartilage,post-operative regions, sexual organs and/or weight supporting areas ofthe feet as well as other conditions of the bones and joints. Thebiomaterials which may be used in the invention may comprise aninjectable composition, preferably in the form of a gel of chitin orchitosan, such as for example a succinochitosan glutamate gel,preferably including particles in suspension in the composition, saidparticles comprising chitin and/or chitosan. The biomaterial of theinvention is bioresorbable, and when particles are in suspension theyare bioresorbable, as well. The resorption time of the gel may bedifferent from the resorption time of the particles. Various medicamentsmay also be used to enhance the treatment of the affected area of thebody.

According to the invention, the use of the biomaterial of the inventionproduces a filling effect, resulting from the injected volume ofcomposition. An important goal of the biomaterial for use in theinvention is to induce fibrosis and tissue formation.

DESCRIPTION OF FIGURES

FIG. 1 is a photo showing the distribution of chitin particles for asuccinochitosan glutamate gel containing 1% chitin particles. The photoof the biomaterial, taken using an OLYMPUS® optical microscope, confirmsthat the particles are distributed homogeneously throughout the gel,naturally remaining in suspension due to the surfactant properties ofchitosan, without the need of additional surfactants.

DETAILED DESCRIPTION OF THE INVENTION

Injecting a high amount of the biomaterial of the invention in a singleinjection may not be the optimal method of treating patients in need ofsaid biomaterial, since the enhancement of the tissue (e.g., dermis) maynot depend on the amount of biomaterial injected in a single injection;it may be preferred to carry out several injections, which may bedistant of a few weeks, for example two months. This embodiment aims atletting the biomaterial almost totally resorb before injecting newbiomaterial.

Fibrosis is induced by biomaterial, which means by the composition andby the particles present in the biomaterial of the invention. When thebiomaterial is injected, it is perceived as foreign bodies and the bodyresponds to this attack by connective tissue hyperplasia, withproliferation of fibroblasts developing from collagen(neo-collagenesis). Fibrosis reaction induced by injecting thebiomaterial of the invention may occur between 15 days and 3 weeks afterinjection.

Inducing fibrosis by injecting the biomaterial, is aimed to createnatural filling tissue which will replace the biomaterial when it isresorbed. It is therefore desirable that the particles, which may beconsidered as being principally responsible for inducing the fibrosis,be resorbed once they no longer fulfill their function of inducingfibrosis, preferably within a period of 1 to 6 months.

Thus, the biomaterial to be used according to the invention, partlybecause of the nature of its composition and partly because of thepresence of particles, proposes a technical solution for patients inneed for implantable filling material, and the product biodegradationand resorption time of the biomaterial may be adapted to the specificneeds of the patients, for example by adjusting the amount of particlesin the composition, thus avoiding the drawbacks of the prior artproducts.

In the present invention, by syringability is meant the ease ofinjection of the biomaterial; syringability generally may be a functionof viscosity and other rheological properties of the biomaterial and ofthe size of the particles included within the biomaterial and ofdiameter of the needle of the syringe. By chitin, is meant a linearpolysaccharide of beta-1.4-N-acetyl-D-glucosamine. By chitosan is meanta linear polysaccharide composed of randomly distributed linkedbeta-1.4-linked N-acetyl-D-glucosamine (acetylated unit) andD-glucosamine (deacetylated unit). The degree of deacetylation ofchitosan may be determined by NMR spectroscopy.

By chitosan derivative, is meant any chitosan salt or acid-derivedchitosan, chitosan glycolate, chitosan lactate, chitosan succinate,hydroxyalkyl chitosan, chitosan acetate, chitosan glutamate and morepreferably succinochitosan glutamate.

According to a preferred embodiment of the invention, the biomaterial tobe used according to the invention comprises or consists of aninjectable bioresorbable polysaccharide composition, preferably in theform of a gel, including resorbable particles in suspension within thecomposition, said particles comprising chitin and/or chitosan.

In one embodiment, the polysaccharide is chitosan or a derivativethereof, preferably having a degree of deacetylation of about 30 toabout 95%, preferably about 70 to about 90%, more preferably about 75 toabout 85%, even more preferably about 80 to about 85%, and mostpreferably about 85%.

Advantageously, the molecular weight of the chitosan or the chitosanderivative of the gel composition or used to make the chitin or chitosanparticles is of about 10 000 to about 500 000 D, preferably about 30 000to about 100 000 D, more preferably about 50 000 to about 80 000 D.

According to an embodiment, the biomaterial includes in its compositionabout 0.1 to about 20%, preferably about 1 to about 20% w/w, morepreferably about 1 to about 12% w/w, even more preferably about 1 toabout 10%, most preferably about 1 to about 5% w/w of polysaccharidewhich is a chitosan or a chitosan derivative. In a specific embodiment,when the polysaccharide is a chitosan derivative, the composition of thebiomaterial may include about 0.1 to about 20% of polysaccharide.According to a particularly preferred embodiment the chitosan derivativeis succinochitosan glutamate.

According to an embodiment, the biomaterial used according to theinvention comprises an injectable bioresorbable polysaccharidecomposition wherein the polysaccharide is succinochitosan glutamate.Advantageously, the succinochitosan glutamate has a degree ofdeacetylation of about 30 to about 95%, preferably about 70 to about90%, more preferably about 75 to about 85%, even more preferably about80 to about 85%, and most preferably about 85%. According to anembodiment, the succinochitosan glutamate has a molecular weight ofabout 10 000 to about 500 000 D, preferably about 30 000 to about 100000 D, more preferably about 50 000 to about 80 000 D. According to anembodiment, the composition comprises about 0.1 to 20%, preferably 1 to10%, more preferably 1 to 5% w/w succinochitosan glutamate by weight ofthe total composition. Advantageously, the biomaterial of the inventionis a gel. The succinochitosan glutamate may be derived from chitosan ofanimal or vegetal origin. Advantageously, the succinochitosan glutamateused to manufacture the biomaterial of the invention is derived fromGMP-grade chitosan.

In another preferred embodiment, the biomaterial is a chitosan orchitosan derivative gel including chitin particles.

According to a most preferred embodiment, the biomaterial to be used isa gel of succinochitosan glutamate, including chitin particles insuspension within the gel.

According to an embodiment, the chitosan used for manufacturing thebiomaterial may be either of animal or vegetal origin. The use of achitosan of animal origin, and more particularly crustaceans (prawnshells) or squids is of economic benefit. The use of a product ofvegetal origin, and more particularly fungal, is generally betterappreciated by consumers. Thus, according to a preferred embodiment, thechitosan used in the biomaterial of the invention, is extracted fromfungi, such as for example Mucoralean strains, Mucor racemosus andCunninghamella elegans, Gongronella butleri, Aspergillus niger, Rhizopusoryzae, Lentinus edodes, Pleurotus sajo-caju, more preferably Agaricusbisporus. According to another embodiment, the chitosan was producedfrom two yeasts, such as, for example Zygosaccharomyces rouxii andCandida albicans.

According to a particular embodiment, the particles included within thebiomaterial to be used according to the invention contain or consistessentially of chitin and/or chitosan which are either of animal orvegetal origin. The particles may also be made of, or include, a mixtureof chitin and chitosan. According to an embodiment, these particles mayconsist solely of chitin or solely of chitosan. According to anembodiment, the chitosan used to make the particles may have a degree ofdeacetylation of about 30 to about 95%, preferably about 70 to about90%, more preferably about 75 to about 85%, even more preferably about80 to about 85%, and most preferably about 85%. Advantageously, thechitosan used to make the particles may be of GMP grade. According to apreferred embodiment, the particles are of chitin obtained byreacetylation of a GMP grade chitosan. According to a preferredembodiment, the biomaterial is essentially free of endotoxins. Accordingto another embodiment, the biomaterial includes deproteinized chitinparticles essentially free of endotoxins.

According to an embodiment, the particles included within thebiomaterial of the invention have a bioresorption time of 1 to 6 months.According to an embodiment, the chitin-only particles with abioresorption time of 1 to 3 months, and chitosan-only particles have abioresorption time of 1 to 4 months.

According to another embodiment, the amount of particles in thebiomaterial of the invention may be of about 0.1 to 10% w/w, preferablyof 1 to 5% w/w, more preferably of 1 to 2% w/w.

The amount of particles included within the biomaterial may depend onthe final inventive application of the biomaterial and of the desiredeffect.

According to a preferred embodiment, the biomaterial is a chitosan orchitosan derivative gel, including 1 to 5% of chitosan particles or 1 to5% of chitin particles. According to another embodiment, the biomaterialis a chitosan derivative gel, including 1 to 2% chitin particles. In aparticularly preferred embodiment, the biomaterial is a gel consistingessentially of a chitosan derivative and water with chitin and/orchitosan particles suspended in the gel, where the gel is essentiallyfree of any other formulation-enhancing agents such as plasticizers,surfactants, viscosity modifiers, and the like.

The biomaterial compositions used according to the invention may alsoinclude certain medicaments such as anesthetics, anti-inflammatories,analgesics, antimicrobials, antibiotics, growth factors, bone repairagents, and other actives and natural products to assist and providefurther advantages in various applications, or may be formulated toexclude such additives.

According to another embodiment, the particles included within thebiomaterial have a mean diameter of about to 150 μm, preferably 5 to 40μm. According to an embodiment, the mean diameter of the particles are 3to 12 μm, and preferably of 5 to 10 μm. According to another embodiment,the mean diameter of the particles are 10 to 32 μm. Preferably, theparticles are microspheres.

Furthermore, these bioresorbable particles in suspension in thebiomaterial should have a diameter such that the syringability of theproduct using 27G (or possibly 30G) needles remains satisfactory.

According to an embodiment, chitin and/or chitosan particles areobtained from chitin or chitosan crystals, having an averagegranulometry at the outset of 200 to 300 μm. The granulometry is reducedby any suitable technique known by one skilled in the art to lower theparticle size of the particles, such as for example, but notlimitatively, spray drying or micronization, optionally repeated morethan once. These particles may then undergo a successive series ofmicronizations, while avoiding cryomicronization which sometimes damagesthe integrity of the micronized molecules. Subsequent sifting stepseliminate those particles which have a granulometry which is either toolarge or too small.

According to an embodiment, the particles of the biomaterial do notcontain polymethacrylic acid and/or ester derivative thereof containinghydroxyl group, polyacrylamide, polymethacrylamide,poly-N-vinyl-2-pyrrolidone, polyvinyl alcohol.

According to another embodiment, the particles are not composite, butmade of a single ingredient, which is preferably chitin.

According to an advantageous embodiment, the biomaterial has a pH whichis compatible with dermatological and internal use, preferably a pHbetween 6.5 and 7.5, and ideally between 6.8 and 7.2.

According to another embodiment, the density of the biomaterial of theinvention is comparable to that of the particles, preferably between0.95 and 1.20, and ideally between 1.00 and 1.10.

The particles may be maintained in suspension by the viscosity of theparticle-containing gel, the natural surfactant effect of chitin andchitosan, and also through the small size of the particles and the factthat their density is more or less equal to that of the gel. Thishomogeneity of density, the surfactant properties of chitin and chitosanand the small particle size ensures satisfactory homogeneity of the gel,avoiding clump formation which may block the fine needles, and avoidingthe need for additional formulation-modifying agents such asplasticizers, surfactants, and viscosity modifiers.

The process for manufacturing a biomaterial to be used in the inventionmay involve steps in which chitosan or chitosan derivative with a degreeof deacetylation of about 30 to about 95%, preferably about 70 to about90%, more preferably about 75 to about 85%, even more preferably about80 to about 85%, and most preferably about 85% is dissolved, followed bysuccessive addition of glutamic acid and then succinic anhydrid, andneutralization. The addition of particles containing chitin and/orchitosan under agitation may be performed at various stages in theprocess, for example before or after addition of glutamic acid or at theend of the process.

During the neutralization step, the pH of this biomaterial is adjustedto somewhere between 6.5 and 7.5, ideally between 6.8 and 7.2, byaddition of a base such as sodium hydroxide or triethanolamine.

The resulting biomaterial may not be affected by either pH ortemperature. The latter is of particular interest as this means theproduct remains stable when stored at room temperature.

The process of manufacturing preferably also includes a sterilizationstep, such as a step involving irradiation or steam sterilization forexample.

According to an embodiment, the chitin and the chitosan used formanufacturing the biomaterial of the invention and/or the particles arefrom one source, which is preferably GMP-grade chitosan. According toanother embodiment, the manufacturing process of the chitin particlessuspended in the biomaterial uses chitin obtained by reacetylation of aGMP-grade chitosan. The chitin used in the manufacturing process of thecomposition and/or of the particles is essentially free of protein.

The invention also relates to a medical device containing the saidbiomaterial for use in treating various conditions. According to aspecific embodiment of the medical device, the said biomaterial isready-to-use in a sterile syringe.

A subject of the invention is a method of treatment to fill a cavity inthe human face or body, which may involve several successive injectionsof the said biomaterial, whereby each injection could be followed bymassage of the surface of the skin over the injection area.

Another subject of the invention is the use of the said biomaterial forrepair and/or treatment of vocal chord insufficiency or other bodilytissue such as cartilage, muscles, ligaments, tendons and/orcorresponding post-operative regions as well as other conditionsaffecting tissue, bones and joints including, but not limited to,ailments due to age, arthritis, and areas of the body havinginsufficient soft tissue (e.g., tailbone, underside of the foot, etc.).

Biomaterial used according to the invention may be used in a method oftreating and/or repairing the loss of fat pads under recurring pressureon the human body comprising administering a composition to the fat padsor surrounding tissue, wherein said composition is in the form of a gelcomprising an injectable bioresorbable polysaccharide, and wherein saidcomposition further comprises resorbable particles which are insuspension in said gel, the said particles comprising chitin and/orchitosan. In one embodiment, the fat pads are located at the soles ofthe feet, at the balls of the feet, at the heels of the feet, or at thecoccyx.

Another subject is the use of the biomaterial as a filler forenhancement or enlargement of male or female genitalia.

The following are further examples of biomaterial which may be used inthe invention. These examples are intended to illustrate possiblebiomaterials for use in the invention in a non-limiting manner:

Example 1 Gel Containing 4% of Chitosan (w/w)

Chitosan, GMP crustacean source, degree of deacetylation 85%, intrinsicviscosity of approx 150 cps (in a 1% acetic acid solution) was dissolvedin purified water. Glutamic acid was added in stoicchiometric quantity(according to DDA) in the solution, which, after 15 to 20 minutes,produced chitosan glutamate. Succinic anhydride was then added (samequantity as glutamic acid) yielding a gel in the form ofsuccino-chitosan glutamate. The pH of the gel was adjusted to 6,8-7,2with sodium hydroxide. Gel was then filtered through a 160 μm filter toeliminate any possible undesired particle. Purified water was then addedso as to obtain a 4% concentration of pure Chitosan in the gel. Gelobtained had a viscosity of approx 2500 cps, and was easy to injectthrough a 30 gauge needle. It was not sensitive to pH or temperature.

Example 2 Gel Containing 2% of Chitosan, in which 1% Chitin Microspheres(w/w) were in Suspension

A gel was prepared in the same way as in example 1, except for theconcentration which was adjusted to 2% of pure CHITOSAN (w/w).Simultaneously, Chitosan was dissolved in a 1% acetic acid solution, andethanol was added in a proportion of 30% of the final solution. A Büchitype spray-dryer was then used in order to obtain Chitosan microspheres,with a granulometry of 5 to 13 μm. These microspheres were poured intoan acetic solution (stoicchiometric quantity of acetic acid calculatedon DDA, so as to be able to reacetylate 25 to 30% of the polymer, so asto obtain more than 50% final acetylation). The resulting chitinmicrospheres were then incorporated into the gel so as to have 1%microspheres (w/w). The final colloidal suspension had a viscosity ofapprox 3500 cps, which made it easy to inject through a 27 gauge needle,and was not sensitive to pH or temperature.

Example 3 Gel Containing 5% of Chitosan, in which 1% Chitin MicronizedParticles (w/w) were in Suspension

A gel was prepared in the same way as in example 1, except for theconcentration which was adjusted to 5% of pure CHITOSAN (w/w). GenuineCHITIN was obtained from the GMP Chitosan supplier. The granulometry ofthis powder was 200-300 μm. The powder was micronized and sieved so asto obtain a powder with a granulometry of 5 to 32 μm. Chitin particleswere then incorporated into the gel so as to have 1% suspension in thegel. The final suspension had a viscosity of approx 4500 cps, and it wasstill possible to inject it through a 27 gauge needle. The final productwas not sensitive to pH or temperature.

1. A method of treating and/or repairing the loss of fat pads underrecurring pressure on the human body comprising administering acomposition to the fat pads or surrounding tissue, wherein saidcomposition is in the form of a gel comprising an injectablebioresorbable polysaccharide, and wherein said composition furthercomprises resorbable particles which are in suspension in said gel, thesaid particles comprising chitin and/or chitosan.
 2. The methodaccording to claim 1, wherein the fat pads are located at the soles ofthe feet, at the balls of the feet, at the heels of the feet, or at thecoccyx.
 3. The method according to claim 1, wherein the compositioncontains one or more medicaments selected from the group of:anesthetics, anti-inflammatories, analgesics, antimicrobials,antibiotics, growth factors or bone repair agents.